The proposed project will characterize a novel signaling system in the pain pathway. VGF (non-acronymic) is a neurosecretory protein similar to the family of chromogranins. It is sorted in the regulated secretory pathway and proteolytically processed into fragments that are released upon stimulation. Currently the precise identity of VGF bioactive fragments and their mechanisms of action are unknown. Our preliminary studies indicate that VGF is rapidly upregulated in models of neuropathic and inflammatory pain. Furthermore, we have found that a VGF-derived peptide (VGF-C30) induces thermal hyperalgesia following spinal administration. These findings have lead us to hypothesize that VGF is involved in pain signaling under conditions of chronic pain. The objective of the proposed project is to establish foundations for in-depth mechanistic analysis of the role of VGF in chronic pain. Specific Aim 1 will evaluate the physiological relevance of VGF in the pain pathway by addressing the following hypotheses: 1) VGF-derived fragments are released from sensory neurons upon stimulation. In these experiments, VGF fragments released from sensory neurons in a depolarization-dependent manner will be characterized using biochemical approaches, including mass spectrometry. 2) The behavioral effects of VGF- C30 are the result of a receptor-mediated activation of an intracellular signaling pathway. The potential receptors activated by VGF-derived bioactive molecules and their signaling mechanisms are completely uncharacterized. The proposed studies will evaluate the binding site(s) of VGF-C30 in spinal cord membrane preparations pharmacologically and biochemically. In addition, based on preliminary evidence, the contribution of mitogen-activated protein kinase pathways to the mechanisms if VGF-C30 signaling in spinal cord will be examined. Specific Aim 2 will address the hypothesis that VGF contributes to mechanisms of chronic pain. The proposed studies will employ two approaches to address this hypothesis: 1) an already established line of VGF knockout mice, and 2) siRNA-mediated knock-down of VGF expression. The combination of these two approaches will allow comprehensive evaluation of the role of VGF in the development and maintenance of chronic pain. In summary, the proposed project will examine the contribution of a novel signaling system to mechanism of chronic pain. The information gained may establish VGF as a potential therapeutic target for chronic pain conditions. The proposed project will characterize a novel signaling system in the pain pathway: the neurosecretory protein VGF (non-acronymic). These studies will lay the foundations for in-depth mechanistic analysis of the role of VGF in chronic pain. The information gained may establish VGF as a potential therapeutic target for chronic pain conditions.